Methodology:
Detailed methods are presented in a manuscript in press at Marine Ecology Progress Series as of 27 June 2022 (Nilsson & Pernet (2022). A brief summary of the larval culture and counting methods are presented below.
Adult echinoids (Dendraster excentricus and Lytechinus pictus) were collected from two sites near San Pedro, California and spawned in lab to yield three full-sibling families per experiment. In each experiment, blastulae were allowed to develop for one day, then distributed to beakers. Ten beakers were prepared for each of three culture density treatments (0.015, 0.05, and 0.25 larvae ml-1). Half (five) of these beakers were allocated a daily ration of Rhodomonas lens (CCMP739) at concentrations of 250 cells ml-1 and the other half (five) allocated 5,000 cells ml-1 to establish two food ration treatments; all received daily water changes.
In each experiment, we assessed the accuracy of larval aliquoting by preparing three additional count control beakers for the 0.05 and 0.25 larvae ml-1 treatments as described above. Larvae in these beakers were fed the higher (5,000 cells ml-1) food ration and cultured at room temperature to accelerate their development, increasing their size and opacity and making them easier to count accurately. At 3 days post fertilization (dpf), the larvae in each count control beaker were concentrated into a small (5 – 20 ml) volume and killed with ethanol. They were then counted to estimate the actual number of blastulae that had been delivered to the experimental beakers at each density. No count control beakers were prepared for the 0.015 larvae ml-1 treatment because those beakers were populated by counting out and pipetting the exact number of desired larvae (15) to each beaker.
Sampling:
The mean larval count for each treatment was compared to the expected number of larvae; as all beakers contained 1000 ml, the expected numbers were 50 and 250 for the 0.05 and 0.25 larvae ml-1 treatments respectively.
These larval counts can be found attached to this metadata record below Data Files within the file, larvae_density_counts.csv.
Analysis:
This analysis was reproduced from Nilsson & Pernet (2022). Studies on feeding structure plasticity in echinoids use diverse statistical approaches in their analyses (McAlister & Miner 2018), a fact that exacerbates the difficulty of comparing results among studies which are already diverse in experimental technique. To enable straightforward comparison with the results of Kacenas & Podolsky (2018), we used the same statistical approach that they did, creating linear mixed-effects models for both dependent variables (PORL and SL) for each experiment. Kacenas & Podolsky (2018) did this using IBM SPSS 24 (IBM Corp. 2016), but we did our analyses in R 4.1.0 (R Core Team 2021) using the lmer function provided by the lme4 1.1-27 package (Bates et al. 2015) and extended by the lmerTest 3.1-3 package (Kuznetsova et al. 2017). Food ration and larval density were treated as fixed effects, beaker as a random effect, body length as a covariate, and either postoral rod length or stomach length as response variables. Estimated marginal means were calculated in R using the emmeans package (Lenth 2021) and compared using the glht function of the multcomp package (Hothorn et al. 2008). To verify that any differences in results were not due to differences between software packages in the implementation of statistical routines, we also analyzed the data from D. excentricus Expt 1 using IBM SPSS 24 (IBM Corp. 2016). Similar values were produced by both packages.
To ensure our statistical conclusions were not particular to the approach that both we and Kacenas & Podolsky (2018) used, we conducted several additional analyses, including similar linear mixed-effects models that included covariate interaction terms as well as a simpler approach using ANOVA to compare PORL and SL adjusted for larval size by dividing each by BL. These analyses, which are detailed in the Supplementary Material, all produced results similar to those of our primary analysis (Tables S1-S5, Fig. S1).
